Process for purifying recombinant human serum albumin

ABSTRACT

The invention provides a process for purifying recombinant human serum albumin (rHSA) by heating a-culture medium containing rHSA and the rHSA-producing host cells, feeding said heated solution upwardly into a fluidized bed in which adsorbent particles are suspended to effect contacting with the adsorbent particles and then recovering the adsorbed fraction containing the rHSA, and a composition comprising rHSA which shows a A350/A280 ratio of below 0.015, when formulated into a 25% solution of said albumin.

This application is a continuation of Ser. No. 08/522089 Aug. 31, 1995now U.S. Pat. No. 5,962,649.

FIELD OF THE INVENTION

This invention relates to a process for easily purifying human serumalbumin obtained in high yields by gene manipulation. The presentinvention also relates to a composition comprising recombinant humanserum albumin which shows an extremely low degree of coloring, which isa serious problem characteristic to recombinant human serum albumin.

BACKGROUND OF THE INVENTION

Human serum albumin (hereinafter referred to simply as HSA) is the mostabundant protein contained in plasma. It contributes to the maintenanceof osmotic pressure in blood and binds to nutrients and metabolites tothereby transport these substances. HSA having these functions has beenemployed as a drug for treating hypoalbuminemia caused by an albuminloss or reduction in albumin synthesis, and in hemorrhagic shock.

HSA has been produced mainly from a fraction of collected blood.However, the process for producing HSA from blood has such problems assporadic supply of blood, economical disadvantage, and contaminationwith undesirable substances such as hepatitis virus. Thus there has beenan urgent requirement to develop a material usable as a substitute fornaturally occurring HSA.

Under these circumstances, techniques for the mass production andpurification of HSA by means of gene manipulation (as a substitute forthe HSA originating in blood) have been developed as recombinant DNAtechnology has progressed.

To purify the HSA obtained by gene manipulation (hereinafter referred toas recombinant HSA and abbreviated as rHSA), it is not appropriate toapply the conventional processes for purifying HSA originating in plasmaas such. This is because the impurities to be eliminated from rHSAcompletely differ from those contained in the HSA originating in plasma.Namely, rHSA is contaminated with, for example, coloring matterscharacteristic to recombinant HSA, proteins originating in the hostcells, polysaccharides, etc. In particular, it is necessary tosufficiently eliminate components originating in the host cells, sincethey are foreign matters for living organisms including human being andthus can cause a problem of antigenicity.

Accordingly, there have been carried out various studies in order toisolate and purify to a sufficient degree rHSA produced via culture fromcomponents originating in the host cells and culture components. One ofthe conventional processes is exemplified by the process which comprisessubjecting a yeast culture medium containing rHSA topressing→ultrafiltration membrane treatment→heating→ultrafiltrationmembrane treatment and then treating by procedures such aschromatography using a cation exchanger and an anion exchanger, andhydrophobic chromatography [JP-A-5-317079 (the term “JP-A” as usedherein means an “unexamined Japanese patent application”) correspondingto EP-A-570916, Biotechnology of Blood Proteins, 1993, 227, 293 -298].Further, the process comprising the above-described procedure followedby the chelate resin treatment or the boric acid/borate treatment hasbeen reported (EP-A-570916, JP-A-6-245789 corresponding to EP-A-612761).

In the above-mentioned conventional process, it is essentially requiredto effect the purification consisting of the above-mentioned severalsteps to thereby eliminate antigens originating in the host cells andachieve a high degree of purification. On the other hand, this processhas such disadvantages as a decrease in the yield of rHSA and aprolonged treating period due to large number of steps. Althoughattempts have been made to elevate the yield of each step so as toimprove the yield of rHSA, it seems that no further improvement in yieldcan be made and thus the yield of rHSA has already reached the upperlimit. Moreover, the conventional process described above suffers fromanother problem that the pressing is effected in an open system and thusthere is a risk of contamination. Namely, hygienic management, which isessentially required in the production of rHSA as a medicine, is highlydifficult therein. In addition, the degree of coloring of rHSA can bereduced only to a A350/A280 ratio of about 0.015 (in the case of asolution containing 250 mg/ml of rHSA) at the lowest (JP-A-7-170993 andJP-A-7-170994 corresponding to EP-A-658569).

On the other hand, there has been developed a process for recovering atarget protein directly from a crude culture medium without affectingany pre-treatment such as elimination of cells or concentration of themedium, after the completion of the cultivation (e.g., the streamlinemethod with the use of expanded bed adsorption technique developed byPharmacia, International Publication in Japan No. 6-500050 correspondingto EP-A-538467).

No case has been reported so far on the application of theabove-mentioned expanded bed adsorption technique to the purification ofrHSA, in particular, the recovery and purification of rHSA from a yeastculture medium. Thus, it remains unknown whether or not such a method isactually useful in the rationalization of the purification of rHSA andthe improvement in the yield of the same. However, it is expected thatthe application of this method or one similar thereto to thepurification of rHSA would contribute to the simplification of theconventional purification treatment consisting of several steps.

However, there arises a problem that, under the acidic conditionsemployed for the adsorption by a streamline column (adsorbent:Streamline SP) for use in the above-mentioned method, rHSA contained inthe culture medium is rapidly degraded by proteases contained in theculture medium and thus the yield of rHSA is seriously lowered. It is,therefore, difficult to apply the above-mentioned expanded bedadsorption technique as such to the purification of rHSA.

Thus, there has been an urgent requirement to develop a process by whichrHSA can be highly purified in a stable state at a high yield withoutspoiling the merits of the expanded bed adsorption technique (i.e., thesimplification and rationalization of the purification process, etc.).

SUMMARY OF THE INVENTION

An object of the present invention is to provide a simple process forpurifying recombinant HSA to a high purity level and to an excellentquality level within a short period of time. Another object of thepresent invention is to provide rHSA from which coloring matterscharacteristic to gene manipulation originating in the host, medium,etc. have been sufficiently eliminated, and a composition comprising theresulting rHSA.

To solve the above-mentioned problems, the present inventors haveconducted extensive studies. As a result, they have found that proteasescan be easily and effectively inactivated by directly heating a culturemedium of an HSA production system in which host cells remain. Based onthis finding, they have further found out that when this heated solutionis contacted directly with adsorbent particles suspended in a fluidizedbed without removing the cells therefrom, rHSA can be easily purified ata high yield. They have furthermore found out that the combination ofthe above-mentioned heating treatment with the adsorbent particletreatment makes it possible to reduce the number of steps of theconventional process for purifying rHSA from five (i.G.,pressing—ultrafiltration membrane treatment→heating→ultrafiltrationmembrane treatment→cation exchanger treatment) to two (heating→adsorbentparticle treatment), thus significantly shortening the purificationperiod while elevating yield.

Moreover, the present inventors have found that their process makes itpossible to obtain rHSA, which is substantially free from any impuritiesoriginating in the host cells and thus exhibits a substantially lowereddegree of coloring compared with the one obtained by the conventionalprocess.

Accordingly, the present invention relates to a process for purifyingrHSA which comprises heating a culture medium containing rHSA and therHSA-producing host, contacting the heated solution with adsorbentparticles suspended in a fluidized bed and recovering the adsorbedfraction. More particularly, it relates to a process for purifying rHSAwhich comprises heating a culture medium containing rHSA and anrHSA-producing host, feeding the heated solution upward into a fluidizedbed in which adsorbent particles are suspended to effect contacting theheated solution with the adsorbent particles, then reversing the flowdirection and feeding downward a buffer to elute and recover the rHSAadsorbed by the adsorbent particles.

The present invention further relates to a purification process whereina culture medium of an rHSA-producing host is heated to a temperature offrom 50 to 100° C. for from 1 minute to 10 hours, a process forpurifying rHSA wherein a heated solution is contacted with adsorbentparticles at a pH value of from about 3 to 5 in an atmosphere of anelectric conductivity of from 0.1 to 50 mS, and a process for purifyingrHSA wherein adsorbent particles are those having a strong cationexchange group.

The present invention further relates to a process for purifying rHSAwherein an adsorbed fraction containing rHSA, which has been recoveredfrom the fluidized bed by the above-mentioned purification process, issubjected to at least one purification treatment selected from a groupconsisting of hydrophobic chromatography, anion exchanger treatment,chelate resin treatment, boric acid/borate treatment and ultrafiltrationmembrane treatment, preferably after heating in the presence of areducing agent.

The present invention furthermore relates to a composition comprisingrecombinant human serum albumin which shows a A350/A280 ratio of below0.015, when formulated into a 25% solution of the albumin (i.e., rHSAconcentration: 250 mg/ml).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph which shows the stabilization effect of the heatingtreatment on an rHSA culture medium.

FIG. 2 is a graph which shows a relationship between the electricconductivity under the atmosphere where the heated solution is contactedwith the adsorbent particles, and the ability of rHSA to bind to theadsorbent particles.

FIG. 3 is a flow chart which shows the optimum flow of the purificationof rHSA from a culture medium (containing yeast cells) with the use ofStreamline SP.

FIG. 4 shows a gel filtration HPLC profile of a Streamline SP eluateincluding the heating step (heating→adsorbent particle) treatment [(a)]and that of a Streamline SP eluate without the heating step (noheating→adsorbent particle) treatment ((b)) (absorbance: measured at 280nm).

FIG. 5 shows a comparison between the conventional process and theprocess of the present invention by monitoring changes in the degree ofcoloring (A350/A280) of rHSA at each step.

FIG. 6 is absorption spectra of rHSA obtained by the process of thepresent invention as compared with rHSA obtained by the conventionalprocess.

FIG. 7 is chromatograms showing the results of GPC-HPLC analysis of rHSAobtained by the process of the present invention (absorbance: measuredat 280 nm).

In these figures, each symbol has the following meaning.

A: control.

B: 68° C., 30 minutes, pH 6.0.

C: 68° C., 30 minutes, pH 6.8.

D: 68° C., 30 minutes, pH 7.5.

E: 68° C., 30 minutes, pH 8.2.

F: 60° C., 2 hours, pH 6.0.

G: 60° C., 2 hours, pH 6.8.

H: 60° C., 2 hours, pH 7.5.

I: 60° C., 2 hours, pH 8.2.

J: 60° C., 2 hours, pH 6.8, 10 mM cysteine.

K: 60° C., 2 hours, pH 7.5, 10 mM cysteine.

L: room temperature (25° C.), 2 hours, pH 6.0.

M: room temperature (25° C.), 2 hours, pH 8.2.

1: rHSA purified by the conventional process(pressing→membrane→heating→membrane→cation exchanger→hydrophobicchromatography→anion exchanger treatment) followed by the chelate resintreatment.

2: rHSA resulting after the anion exchanger (DEAE) treatment inaccordance with the process of Example 1.

3: rHSA resulting after the chelate resin treatment in accordance withthe process of Example 1.

DETAILED DESCRIPTION OF THE INVENTION (1) HSA Obtained by GeneManipulation

In the present invention, the HSA-producing host obtained by genemanipulation is not particularly restricted, so long as it has beenprepared by gene manipulation. Namely, either those which have beenreported in publicly known literatures or those which will be developedin future may be appropriately selected therefor. More particularly,such a host is exemplified by HSA-producing microorganisms obtained bygene manipulation (Escherichia coli, yeasts, Baclillus subtilis, etc.)and animal cells. It is particularly preferable to use yeasts, inparticular, those belonging to the genus Saccharomyces (for example, S.cerevisiae) or Pichia (for example, P. pastoris) as the host. Also,auxotrophic strains and antibiotic-sensitive strains are usabletherefor. It is still preferable to use Saccharomyces cerevisiae AH22strain (a, his 4, leu 2, can 1) or Pichia pastoris GTS115 strain (his4).

Preparation of HSA-producing host, production of rHSA by its culturingand isolation and recovery of rHSA from the cultured broth are allcarried out in accordance with known methods which may be modifiedslightly. For example, preparation of an HSA-producing host may beeffected using a process in which a natural HSA gene is used(JP-A-58-56684 corresponding to EP-A-73646, JP-A-58-90515 correspondingto EP-A-79739 and JP-A-58-150517 corresponding to EP-A-91527), a processin which a modified human serum albumin gene is used (JP-A-62-29985 andJP-A-1-98486 corresponding to EP-A-206733), a process in which asynthetic signal sequence is used (JP-A-1-240191 corresponding toEP-A-329127), a process in which a serum albumin signal sequence is used(JP-A-2-167095 corresponding to EP-A-319641), a process in which arecombinant plasmid is introduced into a chromosome (JP-A-3-72889corresponding to EP-A-399455), a process in which hosts are fused(JP-A-3-53877 corresponding to EP-A-409156), a process in which amutation is generated in a methanol containing medium, a process inwhich a mutant AOX₂ promoter is used (EP-A-506040), a process in whichHSA is expressed in B. subtilis (JP-A-62-215393 corresponding toEP-A-229712), a process in which HSA is expressed in yeast(JP-A-60-41487 corresponding to EP-A-123544, JP-A-63-39576 correspondingto EP-A-248657 and JP-A-63-74493 corresponding to EP-A-251744) and aprocess in which HSA is expressed in Pichia (JP-A-2-104290 correspondingto EP-A-3444S9).

Of these methods, the method in which mutation is induced in amethanol-containing medium is carried out in the following manner. Atransformant of an appropriate host, preferably a Pichia yeast,illustratively a strain GTS115 (NRRL deposition No. Y-15851), isobtained in the usual manner by introducing a plasmid, containing atranscription unit by which HSA is expressed under the control of theAOX₁ promoter, into the AOX₁ gene region of the host (cf. JP-A2-104290). This transformant hardly grows in a medium containingmethanol. In consequence, this transformant is cultured in amethanol-containing medium to generate mutation, and a strain capable ofgrowing in the medium is isolated. Methanol concentration in the mediummay range, for example, from 0.0001 to 5%. The medium may be eithersynthetic or natural. The culturing may be carried out, for example, ata temperature of from 15 to 40° C. for approximately from 1 to 1,000hours.

Culturing of the HSA-producing host may be effected by each of themethods disclosed in the above patents, by a method in which producercells and the product are obtained in high concentrations by a fed-batchculture (a semi-batch culture) which method is carried out by graduallysupplying a high concentration solution of glucose, methanol or the likein appropriate small amounts to avoid high concentration substrateinhibition against the producer cells (JP-A-3-83595) or by a method inwhich the HSA productivity is improved by the addition of fatty acids tothe culture medium (JP-A-4-293495 corresponding to EP-A-504823 and U.S.Pat. No. 5,334,512).

A medium usually employed in the art supplemented with a fatty acidhaving from 10 to 26 carbon atoms or a salt thereof can be used as amedium for culturing a transformed host, and culturing of thetransformant can be carried out under known conditions. The medium maybe either synthetic or natural, but preferably is a liquid medium. Forexample, a suitable synthetic medium may be composed of: carbon sources,such as various saccharides; nitrogen sources, such as urea, ammoniumsalts, nitrates; trace nutrients, such as various vitamins, nucleotides;and inorganic salts, such as of Mg, Ca, Fe, Na, K, Mn, Co and Cu. Anillustrative example of such a medium is YNB liquid medium, whichconsists of 0.7% Yeast Nitrogen Base (Difco) and 2% glucose. Anillustrative example of a useful natural medium is YPD liquid medium,which consists of 1% Yeast Extract (Difco), 2% Bacto Peptone (Difco) and2% glucose. The medium pH may be neutral, weakly basic or weakly acidic.In the case of a methylotrophic host, the medium may be furthersupplemented with methanol in an amount of approximately from 0.01 to5%.

The culturing temperature preferably ranges from 15 to 43° C. (20 to 30°C. for yeast, 20 to 37° C. for bacterium). The culturing period rangesfrom 1 to 1,000 hours, preferably 20 to 360 hours, by means of static orshake culturing or batch, semi-batch or continuous culturing underagitation and aeration. It is desirable to prepare a seed culture priorto the batch culturing by means of static or shake culturing or batch,semi-batch or continuous culturing under agitation and aeration. Theseed culturing may be carried out using the aforementioned YNB liquidmedium or YPD liquid medium, preferably at 30° C. (for yeast) or 37° C.(for bacterium) and for 10 to 100 hours.

(2) Purification of rHSA

(i) Heating Treatment

In the process for purifying rHSA according to the present invention,the culture medium of the HSA-producing host obtained in theabove-mentioned cultivation step can be directly heat-treated whilecontaining the host cells and without effecting any separation proceduresuch as centrifugation or ultrafiltration membrane treatment. That is tosay, the heating treatment is carried out at a first step in thepurification process of the present invention.

Heating is performed usually at 50 to 100° C. for 1 minute to 10 hours,preferably at 60 to 80° C. for 20 minutes to 3 hours and stillpreferably at 68° C. for 30 minutes. It is preferable to perform thistreatment in the presence of a stabilizer. Examples of the stabilizerinclude acetyltryptophan, organic carboxylic acids having 6 to 18 carbonatoms and salts thereof. These stabilizers may be used together.Acetyltryptophan is added in such an amount as to give a finalconcentration of, for example, about 1 to 100 mM. Examples of theorganic carboxylic acids having 6 to 18 carbon atoms include caproicacid, caprylic acid, capric acid, lauric acid, palmitic acid and oleicacid. It is preferable to use 10 mM of caprylic acid. Examples of thesalts thereof include alkali metal salts (sodium salt, potassium salt,etc.) and alkaline earth metal salts (calcium salt, etc.). These organiccarboxylic acids having 6 to 18 carbon atoms or salts thereof may beadded in an amount of, for example, about 1 to 100 mM.

To suppress the coloration caused by heating, it is effective to addabout 1 to 100 mN, preferably 5 to 30 mM, of a thiol compound (e.g.,mercaptoethanol, cysteine, reduced glutathione, etc.) and, preferably tofurther add 10 to 1,000 mM of aminoguanidine in the heating step(JP-A-3-103188).

In the conventional process, either a supernatant (filtrate) or cellsobtained by centrifuging or filtering a host culture medium are heated.This is because it is feared that when a culture medium containing cellsis directly heated, the leakage of impurities, lipids, nucleic acids,proteases, etc. would exert undesirable effects on the purification ofthe target substance. Thus, it has been unknown whether direct heatingof a culture medium containing cells is effective for purification ofthe target substance or not.

According to the present invention, however, it has been revealed thatwhen the culture medium containing the rHSA and the host cells isdirectly heated, neither the structural function of rHSA nor the yieldthereof is deteriorated but the powerful proteases contained in theculture medium can be effectively inactivated. Thus the process for theinactivation of the proteases can be simplified. Moreover, HSA obtainedby gene manipulation can be effectively purified by the process as willbe described hereinafter.

(ii) Dilution of Heated Solution and Adjustment of its Properties

The heated solution obtained in the above (i) is then treated withadsorbent particles suspended in a fluidized bed. Prior to thistreatment, it is preferable to dilute the heated solution so as tocontact this heated solution with the adsorbent particles in anatmosphere of an electric conductivity of from 0.1 to 50 mS, preferablyfrom 0.5 to 35 mS and still preferably from 5 to 15 mS. As will bedescribed in Test Example 3 hereinafter, the amount of rHSA binding tothe adsorbent particles increases as the dilution of the heated solutionis elevated and the electric conductivity of the atmosphere, at whichthe heated solution is contacted with the adsorbent particles, islowered, and attains the maximum level at an electric conductivity ofaround 8 to 12 mS. The solvent to be used as the diluent is notparticularly restricted, so long as the structural function of rHSA inthe heated solution is not thereby deteriorated. It may be appropriatelyselected while taking the adsorption conditions into consideration. Thediluent is exemplified by an acetate buffer of a concentration of 50 mMor below and by distilled water. From the viewpoint of convenience, itis preferable to use distilled water.

Next, the pH value of the solution is regulated to an acidic level,which is suitable for the adsorption by the adsorbent particles. It isadjusted to usually from pH 3 to 5, preferably from pH 4 to 4.8 and morepreferably about pH 4.5. Although any acidic solution may be used forthe regulation of pH without restriction, it is preferable to use aceticacid.

(iii) Adsorbent Particle Treatment

Following the dilution and adjustment of the pH value, the resultingheated solution is contacted with the adsorbent particles.

Examples of the adsorbent particles include a carrier having a cationexchange group (i.e., a cationic adsorbent), such as adsorbent particlesof the sulfo group type or carboxyl group type. Adsorbent particles ofthe sulfo group type are exemplified by sulfo-agarose (Streamline SP,S-Sepharose, both manufactured by Pharmacia), sulfo-cellulose(S-Cellulofine, manufactured by Chisso Corporation), sulfopropyl-agarose(SP-Sepharose, manufactured by Pharmacia), SP-Cellthru-Big Beads(manufactured by Sterogene), sulfopropyl-dextran (SP-Sephadex,manufactured by Pharmacia) and sulfopropyl-polyvinyl (SP-Toyopearl,manufactured by Tosoh Corporation). Adsorbent particles of the carboxylgroup type are exemplified by carboxymethyl-agarose (CM-Cellthru-BigBeads, manufactured by Sterogene), carboxymethyl-dextran (CM-Sephadex,manufactured by Pharmacia) and carboxymethyl-cellulose (CM-Cellulofine,manufactured by Chisso Corporation). It is preferable to use stronglycationic adsorbent particles of the sulfo group type, with Streamline SP(manufactured by Pharmacia) particularly preferred.

The particle size of tho adsorbent particles usually ranges from, forexample, 30 to 1,100 μm, preferably from 100 to 300 μm.

The contact can be effected at a pH value of from about 3 to 5,preferably from about 4 to 4.8 and more preferably about 4.5, and at asalt concentration of from about 0.01 to 0.2 M, preferably from about0.05 to 0.1 M.

It is preferable to preliminarily equilibrate the adsorbent particlesunder such contact conditions as described above. More particularly, itis preferable to equilibrate the adsorbent particles with a 50 mMacetate buffer (pH 4.5) containing 50 mM of sodium chloride.

The adsorbent particles are usually equilibrated and the sample isinjected into the fluidized bed containing the adsorbent particles,contacted with the adsorbent particles and then eluted from thefluidized bed in accordance with the following procedure.

Namely, the above-mentioned adsorbent particles are first packed into anappropriate column and allowed to sink. Then an equilibration buffer isfed upward from the bottom port of the column to thereby suspend theadsorbent particles. In this step, the flow rate of the buffer flowingupward in the column serves as the counterbalance to the adsorbentparticles sedimenting due to gravity and thus the adsorbent particlesare uniformly suspended in an equilibrated state (i.e., a so-calledfluidized bed). Into the column, in which the above-mentioned fluidizedbed has been formed, the crude culture medium containing the cells,which has been treated according to the above (i) heating and (ii)dilution steps, is supplied upwardly from the bottom port of the column.Then the target rHSA binds to the adsorbent particles, while fineparticles and impurities originating in the host cells or the culturemedium pass as such among the adsorbent particles in the fluidized bedand are thus discarded from the top port of the column. Also, impuritiesloosely binding to the adsorbent particles are washed away with thewashing buffer which is successively and upwardly supplied. It ispreferable that these procedures are performed in accordance with theexpanded bed adsorption technique [Journal of Chromatography, 597(1992), 129-145].

As the washing buffer, an equilibration buffer is employed.

The target protein, i.e., rHSA is recovered by reversing the flowdirection and feeding downwardly an elution buffer from the top port ofthe column. Elution of rHSA can be carried out at the pH value rangingfrom about 8 to 10, preferably from about 8.5 to 9.5 and more preferablyabout 9, and at the salt concentration ranging from about 0.2 to 0.5 M,preferably from about 0.3 to 0.4 M. A particular example of the elutionbuffer is a 0.1 M phosphate buffer (pH 9) containing 0.3 M of sodiumchloride.

The above-mentioned operations including equilibration of the adsorbentparticles, injection of the sample into the fluidized bed containing theadsorbent particles, contact with the adsorbent particles, elution fromthe fluidized bed, etc. can be easily and efficiently carried out at ahigh reproducibility by using a Streamline System (manufactured byPharmacia) provided with a Streamline column (adsorbent: Streamline SP,manufactured by Pharmacia).

Thus rHSA having a high purity can be obtained. The purity of the rHSAobtained by the above-mentioned treatment with the adsorbent particlesis almost comparable to that of the rHSA obtained by the conventionalprocess consisting of several steps including pressing→ultrafiltrationmembrane treatment→heating→ultrafiltration membrane treatment→cationexchanger treatment. Moreover, rHSA can be obtained in a stable form(i.e., without being degraded) at a high yield owing to the heating ofthe culture medium prior to the treatment with the adsorbent particles.Accordingly, the present invention makes it possible to reduce thenumber of steps of the above-mentioned conventional process forpurifying rHSA from five to two and thus largely shorten thepurification period. The present invention further makes it possible toelevate the yield of rHSA from the culture medium, compared with theconventional process.

The rHSA obtained by these treatments can be further purified by aconventional purification process. Examples of the purification processto be used herein include those commonly employed in the art such ashydrophobic chromatography, ultrafiltration membrane treatment, anionexchanger treatment, chelate resin treatment and boric acid/boratetreatment. Either one of these treatments or a combination thereof maybe used. In order to obtain a purified rHSA product of improvedqualities, it is preferable to perform the hydrophobic chromatography,ultrafiltration membrane treatment, anion exchanger treatment,ultrafiltration membrane treatment, chelate resin treatment, boricacid/borate treatment and ultrafiltration membrane treatment in thisorder.

Prior to the above-mentioned purification, it is preferable to heatagain the adsorbed fraction eluted after contacting the solution withthe adsorbent particles in the presence of a reducing agent. As will beshown in Test Example 6 hereinafter, this heating treatment is highlyeffective in lowering the degree of coloring characteristic of rHSA,though the yield of rHSA is not affected thereby as compared with thecase that this heat treatment is omitted. Namely, this heating treatmentmakes it possible to significantly eliminate the coloring matters by thesubsequent purification procedure.

The heating temperature is usually from 50 to 100° C., preferably fromabout 60 to 80° C. and more preferably 60° C. The heating period isusually from 10 minutes to 10 hours, preferably from about 30 minutes to5 hours and more preferably 1 hour.

The reducing agent to be used herein is not particularly restricted, solong as it has a reducing effect. Examples thereof include low molecularweight compounds having an SH group (e.g., cysteine, cysteamine,cystamine, methionine, glutathione, etc.), sulfites, pyrosulfites,phosphorous pyrosulfites and ascorbic acid. Cysteine is preferably used.Regarding the addition level, for example, cysteine may be added in suchan amount as to give a final concentration of about 1 to 100 mM, while asulfite may be added in such an amount as to give a final concentrationof about 0.001 to 10%.

It is preferable to perform this treatment in the presence of astabilizer. Examples of the stabilizer include acetyltryptophan andorganic carboxylic acid having 6 to 18 carbon atoms or salts thereof.These stabilizers may be used together. Acetyltryptophan may be added,for example, in such an amount as to give a final concentration in thesolution of about 1 to 100 mM. Examples of the organic carboxylic acidshaving 6 to 18 carbon atoms include caproic acid, caprylic acid, capricacid, lauric acid, palmitic acid and oleic acid. It is preferable to use10 mM of caprylic acid. Examples of the salts thereof include alkalimetal salts (e.g., sodium salt, potassium salt, etc.) and alkaline earthmetal salts (e.g., calcium salt, etc.). These organic carboxylic acidshaving 6 to 18 carbon atoms or salts thereof may be added in an amountof, for example, about I to 100 mM. By further adding 10 to 1,000 mM ofaminoguanidine, the coloration due to heating can be suppressed(JP-A-3-103188).

(a) Hydrophobic Chromatography

The hydrophobic chromatography can be performed in a conventionalmanner. Examples of the carrier for hydrophobic chromatography includeinsoluble carriers having an alkyl group carrying 4 to 18 carbon atoms(butyl group, octyl group, octyldecyl group, etc.) or a phenyl group. Itis preferable to use one having a phenyl group such as phenyl-cellulose(Phenyl-Cellulofine, manufactured by Chisso Corporation). In this step,rHSA can be recovered into the unadsorbed fraction. The contact may beperformed, for example, at about pH 6 to 8, preferably at about pH 6.5to 7, and at a salt concentration of about 0.01 to 0.5 M, preferablyabout 0.05 to 0.2 M.

(b) Anion Exchanger Treatment

The anion exchanger treatment may also be performed in a conventionalmanner. Any anion exchanger may be used, so long as it is an insolublecarrier having an anion exchange group. Examples of the anion exchangegroup include a diethylaminoethyl (DEAE) group and a quaternaryaminoethyl (QAE) group. It is preferable to use one having a DEAE groupsuch as DEAE-agarose (DEAE-Sepharose, manufactured by Pharmacia),DEAE-dextran (DEAE-Sephadex, manufactured by Pharmacia) andDEAE-polyvinyl (DEAE-Toyopearl, manufactured by Tosoh Corporation). Inthis step, rHSA can be recovered into the unadsorbed fraction. Thecontact may be performed, for example, at about pH 6 to 8, preferably atabout pH 6.5 to 7, and at a salt concentration of about 0.01 to 0.1 M.

By this anion exchanger treatment, coloring matters and trace impuritiescan be eliminated.

(c) Ultrafiltration Membrane Treatment

After the completion of the hydrophobic chromatography and/or the anionexchanger treatment, the rHSA-containing fraction thus recovered ispreferably subjected to the ultrafiltration membrane treatment. By thisultrafiltration membrane treatment, pyrogens can be eliminated. In theultrafiltration membrane treatment, it is preferable to use anultrafiltration membrane having a molecular weight cutoff of from 100 to300 R. As a particular example thereof, a Pellicon cassette membrane100K (manufactured by Millipore) may be cited.

(d) Chelate Resin Treatment

The chelate resin treatment is effective particularly in eliminatingcoloring matters which are characteristic to HSA obtained by genemanipulation. In the above-mentioned purification process, thistreatment is preferably carried out following the hydrophobicchromatography→ultrafiltration membrane treatment→anion exchangertreatment→ultrafiltration membrane treatment. It is performed bycontacting a chelate resin having a specific ligand with rHSA and therHSA is obtained in the passed fraction. It is preferable that thecarrier part of the chelate resin is one having hydrophobic properties.Examples thereof include a styrene/divinylbenzne copolymer and anacrylic acid/methacrylic acid copolymer. On the other hand, examples ofthe ligand part of the chelate resin include polyol groups such asN-methylglucamine, polyamine groups (including polyalkylene polyaminessuch as polyethylene polyamine) having plural imino groups, aminogroups, ethyleneimino groups, etc. in the molecule and thiourea groups.It is convenient to use commercially available those having astyrene/divinylbenzene copolymer carrier, such as DIAION CRB02 (ligand:N-methylglucamine group, manufactured by Mitsubishi Kasei Corporation),LEWATIT TP214 (ligand:—NHCSNH₂, manufactured by Bayer) and AmberliteCG4000.

Appropriate conditions for this chelate resin treatment are as follows.

pH: acidic or neutral or basic (pH 3 to 9, preferably pH 4 to 7).

Period: 1 hour or longer, preferably 6 hours or longer.

Ionic strength: 50 mmho or below, preferably from 1 to 10 mmho.

Mixing ratio: 0.1 to 100 g, preferably 1 to 10 g (wet basis) of resinper 250 mg of HSA.

(e) Boric Acid/Borate Treatment

By further treating the rHSA-containing solution, which has beenobtained by the above-mentioned treatment, with boric acid or a borate(referred to as boric acid/borate herein), impurities having anantigenicity originating in the host and nonantigenic free impuritiesdetectable by the phenol-sulfuric acid method can be eliminated.

Examples of the boric acid usable herein include orthoboric acid,metaboric acid and tetraboric acid. Examples of the borate includealkali metal salts (e.g., sodium salt, potassium salt, etc.) andalkaline earth metal salts (e.g., calcium salt, etc.). It is preferableto use calcium tetraborate. The boric acid or borate may be added insuch an amount as to give a final concentration of from about 0.01 to 1M, preferably from about 0.05 to 0.2 M. This treatment is performed, forexample, at about pH 8 to 11, preferably about pH 9 to 10, for about 1to 100 hours, preferably about 5 to 50 hours. In this step, therHSA-containing solution having a low electric conductivity is moredesirable. For example, the electric conductivity of the rHSA-containingsolution is 1 mS or below. Also, the rHSA-containing solution having ahigh rHSA concentration is more desirable. For example, the rHSAconcentration is 5% or more, preferably from about 15 to 25%.

After the completion of the above-mentioned boric acid/borate treatment,rHSA is recovered from the supernatant by a conventional method such ascentrifugation or ultrafiltration.

(3) Properties of Highly Purified HSA Originating in Gene Manipulation

The highly purified rHSA thus obtained is a homogeneous substance havinga molecular weight of about 67,000 and an isoelectric point of 4.9. Itconsists of monomers alone, being substantially free from dimers,polymers or decomposition products (molecular weight: about 43,000).Also it is substantially free from any antigenic impurities orpolysaccharides originating in the producer host. When formulated intoan rHSA solution of 250 mg/ml (a 25% solution), it has a A350/A280 ratioof below 0.015, preferably 0.013 or below and more preferably from about0.01 to 0.015. rHSA having such reduced degree of coloring (i.e., a lowA350/A280 value) can be obtained by using an appropriate combination ofthe known purification techniques [the above-mentioned techniques (a) to(e), etc.].

The present invention makes it possible for the first time to providerecombinant HSA (or a composition containing the same) showing aA350/A280 ratio of below 0.015, when formulated into a 25% solution ofrHSA.

(4) Formulation

The rHSA obtained by the process of the present invention can beformulated into preparations by known methods (ultrafiltration membranetreatment, addition of stabilizer, antiseptic filtration, pipetting,freeze-drying, etc.). The rHSA preparations thus obtained may be appliedto clinical purposes as serum albumin preparations in the same dose andin the same manner as those employed in the case of the conventional HSAoriginating in plasma. They are also usable as stabilizers, fillers orcarriers for drugs.

The term an “rHSA-containing composition” as used herein means amaterial which contains the high purity rHSA of the present invention ata high concentration but lower than 100% together with othercomponent(s) in a trace amount.

To further illustrate the present invention in greater detail, and notby way of limitation, the following Examples will be given.

EXAMPLE 1 (1) Heating Treatment of Culture Medium

An HSA-producing yeast Pichia pastoris was acquired and incubated inaccordance with the method described in EP-A-655503.

About 2.8 liter of the culture medium including cells thus obtained washeated to 68° C. for 30 minutes as such. The heating treatment wasperformed in the presence of 10 mM of sodium caprylate. This culturemedium had a pH value of 6. Next, the heated solution was quickly cooledto about 15° C. and diluted about 2-fold with distilled water (totalvolume 5.5 liter). Then the pH value thereof was regulated to 4.5 withan acetic acid solution.

(2) Adsorbent Particle Treatment (Streamline SP Treatment)

To a Streamline SP column (C50, 5×100 cm, gel volume; 300 ml,manufactured by Pharmacia), which had been equilibrated with a 50 mMacetate buffer (pH 4.5) containing 50 mM of sodium chloride, was fedupwardly 5.5 liter of the culture medium (electric conductivity: <10 mS)containing the yeast cells which had been obtained by theabove-mentioned heating treatment (1). The feeding was made at a flowrate of 100 cm/h under stirring. Next, the same buffer (2.5 times byvolume as much as the column capacity) as the one employed for theequilibration of the column was fed upwardly into the column to therebywash the column at a flow rate of 100 cm/h for 1 hour and then at 300cm/h for 30 minutes. Subsequently, the flow direction was reversed andan eluent [a 100 mM phosphate buffer (pH 9) containing 300 mM of sodiumchloride, flow rate: 50 cm/h] was fed into the column. Thus a fractioncontaining rHSA was obtained.

The rHSA-containing fraction thus eluted was detected by measuring theabsorbance at 280 nm.

(3) Heating Treatment

The rHSA-containing fraction thus obtained was heated at 60° C. for 1hour in the presence of 10 mM of cysteine, 5 mM of sodium caprylata and100 nM of aminoguanidine hydrochloride at pH 7.5.

(4) Hydrophobic Chromatography

The rHSA solution heated in the above (3) was poured into a columnpacked with Phenyl-Cellulofine (5×25 cm, gel volume: 500 ml,manufactured by Chisso Corporation) which had been equilibrated with a50 mM phosphate buffer (pH 6.8) containing 0.15 M of sodium chloride.Under these conditions, the rHSA was not adsorbed by thePhenyl-Cellulofine column but passed therethrough. The rHSA-containingsolution passing through the column was concentrated to a volume ofabout 0.2 liter using an ultrafiltration membrane having a molecularweight cutoff of 30,000 (manufactured by Millipore) and therHSA-containing solution was replaced by a 50 mM phosphate buffer (pH6.8).

(5) Anion Exchanger Treatment

After the completion of the hydrophobic chromatography, therHSA-containing solution, which had been concentrated andbuffer-replaced, was poured into a column packed with DEAE-Sepharose FF(5×25 cm, gel volume: 500 ml, manufactured by Pharmacia) which had beenequilibrated with a 50 mM phosphate buffer (pH 6.8). Under theseconditions, the rHSA was not adsorbed by the DEAE-Sepharose column butpassed therethrough. The rHSA passing through the column wasconcentrated to a volume of about 0.2 liter using an ultrafiltrationmembrane having a molecular weight cutoff of 30,000 (manufactured byMillipore) and the rHSA-containing solution was replaced by distilledwater.

(6) Chelate Resin Treatment

To 0.2 liter of the purified rHSA having a concentration of about 7% wasadded acetic acid to thereby regulate the pH value to 4.5. Then it waspoured into a column packed with DIAION CRB02 (5×2.5 cm, gel volumes 500ml, manufactured by Mitsubishi Kasei Corporation), which had beenequilibrated with a 50 mM sodium acetate buffer (pH 4.5), and circulatedovernight. Under these conditions, the rHSA was not adsorbed by the gelbut passed through the column.

(7) Boric Acid/Borate Treatment

The rHSA concentration was adjusted to 2.5%, while the electricconductivity of the solution was regulated to 1 mS or below. Sodiumtetraborate was added thereto to give a final concentration of 100 mM.Next, calcium chloride was added thereto to give a final concentrationof 100 mM, while maintaining the pH value at 9.5. After allowing tostand for about 10 hours, the precipitate thus formed was removed andthe supernatant was recovered, concentrated and desalted. Then it wasconcentrated by using an ultrafiltration membrane having a molecularweight cutoff of 30,000 (manufactured by Millipore) and subjected tobuffer replacement. If necessary, stabilizers (sodium caprylate andacetyltryptophan) were added followed by filter sterilization using a0.22 μm filter (manufactured by Millipore). The resulting rHSA solutioncan be used for injection.

TEST EXAMPLE 1 Stabilizing Effect of Heating Treatment on rHSA CultureMedium

It is known that potent proteases contained in an rHSA culture mediumdegrade rHSA. As Table 1 shows, the degradation of rHSA proceedsextremely quickly under acidic conditions of pH 4.

TABLE 1 pH stability of rHSA culture medium Storage Storage HSA conc.Yield Sample Condition pH value (mg/ml) (%) culture medium 15° C., 15 hrcontrol 7.6 100.0 (containing (stationary) yeast cells) 6.0 7.7 100.94.5 7.2 94.0 4.0 3.0 39.9

rHSA was adsorbed by Streamline SP at about pH 4.5. Thus the stabilityof rHSA at about pH 4.5 was examined and heating conditions effectivefor inactivation of proteases as a pre-treatment for maintaining rHSA ina stable state were examined by determining the stability of rHSA afteradjusting the pH value of the heated solution to 4.5 and allowing it tostand at room temperature overnight. FIG. 1 shows the results. In theheating stop, sodium caprylate was added to each sample to give a finalconcentration of 10 mM. The heating conditions employed are as follows.

A: control.

B: 68° C., 30 minutes, pH 6.0.

C: 68° C., 30 minutes, pH 6.8.

D: 68° C., 30 minutes, pH 7.5.

E: 68° C., 30 minutes, pH 8.2.

F: 60° C., 2 hours, pH 6.0.

G: 60° C., 2 hours, pH 6.8.

H: 60° C., 2 hours, pH 7.5.

I: 60° C., 2 hours, pH 8.2.

J: 60° C., 2 hours, pH 6.8, 10 mM of cysteine.

K: 60° C., 2 hours, pH 7.5, 10 mM of cysteine.

L: room temperature (25 7° C.)1 2 hours, pH 6.0.

M: room temperature (25° C.), 2 hours, pH 8.2.

As a result, it was more effective to heat to 68° C. for 30 minutes thanto heat to 60° C. for 2 hours. Regarding the pH value, the mostdesirable results were obtained at around pH 6, which was the initial pHvalue of the culture medium to be heated.

TEST EXAMPLE 2 Relationship Between pH Value of rHSA Culture Medium andBinding Ability to Adsorbent

The pH value of the culture medium (rHSA: 55.6 mg) was adjusted tovarious levels (pH 4.5 to 4.9) with acetic acid followed by the additionof 1 ml of a Streamline SP gel equilibrated with a 50 mM acetate buffer.After mixing and stirring at room temperature for 1 hour and washingwith each equilibration buffer, the amount (%) of the rHSA remaining inthe unadsorbed fraction was determined. As a result, it was found outthat the amount of the rHSA binding to the adsorbent increased with adecrease in the pH value and attained the maximum at around pH 4.5(Table 2).

TABLE 2 pH value of rHSA culture medium and binding ability to gelSample (adsorption rHSA in unadsorbed rHSA in unadsorbed condition)fraction (mg) fraction (%) starting material 55.6 100.0 no gel added: pH4.5 51.0 91.7 1 ml of gel added: pH 4.5 5.1 9.2 pH 4.6 7.6 13.7 pH 4.714.9 26.8 pH 4.8 33.1 59.5 pH 4.9 49.4 88.8

TEST EXAMPLE 3 Relationship Between Electric Conductivity of Atmospherefor the Contact of Heated Solution With Adsorbent Particles and BindingAbility of rHSA to Adsorbent Particles

After heating, the culture medium [electric conductivity: about 20 mS(at 25° C.), rHSA: 47.1 mg] was diluted with distilled water to givevarious dilutions and the pH value of each dilution was regulated to 4.5with acetic acid followed by the addition of 1 ml of a Streamline SP gelwhich had been equilibrated with a 50 mM acetate buffer (pH 4.5)containing 50 mM of sodium chloride. After mixing at room temperaturefor 1 hour and washing with the equilibration buffer, rHSA was elutedwith a 0.1 M phosphate buffer (pH 9) containing 0.3 M of sodiumchloride. As a result, it Was found out that the amount of rHSA bindingto the adsorbent particles increased with an increase in the dilutionand a decrease in the electric conductivity of the solution and attainedthe maximum level at the electric conductivity of the atmosphere (in thegel mixture), in which the heated solution was contacted with theadsorbent particles, of about 8 to 12 mS (FIG. 2).

TEST EXAMPLE 4 Stability of Streamline SP Eluate

The culture medium was diluted 2-fold with distilled water and the pHvalue was adjusted to 4.5 with acetic acid. Then a definite amount of aStreamline SP gel, which had been equilibrated with a 50 mM acetatebuffer (pH 4.5) containing 50 mM of sodium chloride, was added theretoand the mixture was stirred at room temperature for 1 hour. Next, thegel was washed with the equilibration buffer and rHSA was eluted byusing a 0.1 M phosphate buffer (pH 9) containing 0.3 M of sodiumchloride. Then the stability of the rHSA in the fraction (pH 9) wasexamined. As a result, the rHSA in the culture medium which had beenpreliminarily neater (68° C., 30 minutes) showed no change after 3 days,while the rHSA originating in the untreated culture medium was reducedto about 50% due to degradation (Table 3).

TABLE 3 Stability of Streamline SP eluate (room temperature, 3 days, pH8) rHSA before rHSA after Residual storage storage rate Sample (mg/ml)(mg/ml) (%) eluate from unheated 4.39 2.44 55.7 rHSA culture eluate fromheated 4.55 4.45 97.6 rHSA culture (60° C., 2 hr) eluate from heated4.52 4.67 103.4 rHSA culture (68° C., 30 min)

TEST EXAMPLE 5 Comparison of rHSA Yield and Degree of Coloring Between(Heating→Adsorbent Particle) Treatment and (no Heating→AdsorbentParticle) Treatment

Based on the results of Test Examples 1 to 4, an optimum flow of theheating→adsorbent particle treatment process was established (FIG. 3).

In accordance with the flow of FIG. 3, an attempt was made to purifyrHSA from a culture medium (2.8 liter) containing yeast cells using aStreamline SP column (5×100 cm, gel volume: 300 ml). On the other hand,a culture medium (2.7 liter) containing yeast cells was subjected to thetreatment with the adsorbent particles without initially heating tothereby purify rHSA.

As a result, the total yield achieved by the (no heating→adsorbentparticle) treatment was 50%, while a higher total yield (about 85%) wasachieved by the (heating→adsorbent particle) treatment of the presentinvention. Also, the rHSA obtained by the (no heating→adsorbentparticle) treatment showed a degree of coloring of 0.048 in terms ofA350/A280, while the rHSA obtained by the (heating→adsorbent particle)treatment of the present invention showed a lower degree of coloring(0.0345) (Table 4). FIG. 4 shows a comparison between a gel filtrationHPLC profile of an Streamline eluate in the (heating→adsorbent particle)treatment and that of an Streamline SP eluate in the (noheating→adsorbent particle) treatment. The latter shows seriousdecomposition and degradation of rHSA.

TABLE 4 Purification of rHSA by Streamline SP column Degree of VolumerHSA Yield coloring Sample (liter) (g) (%) (A350/A280) heated sample:culture medium 2.8 19.3 100 0.0475 heated (68° C., 30 min) 5.5 17.5 90.50.0291 column pass 10.5 1.3 6.6 — column eluate 1.2 16.5 85.4 0.0345unheated sample: culture medium 2.7 11.9 100 0.0482 solution added to5.3 10.0 84.2 0.0366 column (diluted, pH-adjusted) column pass 10.5 1.08.8 — column eluate 1.2 5.9 50.0 0.0480

TEST EXAMPLE 6 Effect of Heating With Cysteine on Degree of Coloring ofrHSA

The rHSA eluate from the Streamline SP column in the (heating→adsorbentparticle) treatment of the above Table 4, which was used as a startingmaterial, was subjected to the hydrophobic chromatography and anionexchanger treatment described in Example 1 (4) and (5) and thus thedegree of coloring (A350/A280) was evaluated. In this evaluation, twosamples of the Streamline SP eluates, i.e., one resulted from heating inthe presence of cysteine (final concentration: 10 mM) and the otherresulted from non-heating in the presence of cysteine, were employed. Asa result, the rHSA yields of these two samples were almost the sameregardless of the presence of cysteine. Regarding the degree ofcoloring, however, the sample which had been heated in the presence ofcysteine showed 0.0128, which was significantly lower compared with0.0184 of the unheated sample, after the anion exchanger treatment(Table 5).

TABLE 5 Purification of rHSA after the Streamline step Degree ofcoloring Sample (A350/A280) Streamline eluate 0.0311 heated withcysteine 0.0212 phenyl-treated 0.0197 ultrafiltered (UF30K-R) 0.0205DEAE-treated 0.0128 Streamline eluate 0.0311 phenyl-treated 0.0270ultrafiltered (UF30K-R) 0.0275 DEAE-treated 0.0184

TEST EXAMPLE 7 Reduction in Degree of Coloring (A350/A280) of rHSA byIntroducing Streamline SP Treatment

FIG. 5 shows a comparison of changes in the degree of coloring(A350/A280) between rHSA at each stop in the conventional process andrHSA in a process of the present invention, which included theStreamline SP step, until the step of the anion exchanger treatment. Inthe process or the present invention wherein the Streamline SP treatmentwas employed and heating was effected immediately thereafter in thepresence of cysteine, a large difference from the conventional processwas observed at the step of hydrophobic chromatography. After thecompletion of the anion exchanger treatment, an extremely low degree ofcoloring (0.0128) was observed. FIG. 6 shows a comparison of theabsorption spectrum between a sample resulting after the chelate resintreatment following the conventional process as shown in FIG. 5 (curve1) and samples resulting after the anion exchanger treatment and thechelate resin treatment of the purification process of the presentinvention as described in Example 1 (curve 2 and 3). As a result, theabsorption spectrum of the rHSA purified by the process of the presentinvention already showed a remarkably low pattern over the visibleregion (350-700 nm) after the anion exchanger treatment, compared withthe sample resulting after the chelate resin treatment following theconventional process as shown in FIG. 5. After treating with the chelateresin in the inventive process, this difference became more obvious.

TEST EXAMPLE 8 Analysis of rHSA Resulting From the Process of thePresent Invention by Gel Filtration HPLC

FIG. 7 shows the results of gel filtration high performance liquidchromatography (GPC-HPLC) analysis of the rHSA samples which resultedafter stated steps of the process of the present invention as describedin Example 1 (rHSA culture medium, Streamline SP eluate, Streamline SPunadsorbed fraction, DEAE-post-treated fraction). As a result, it wasclarified that high molecular weight substances other than rHSA and lowmolecular weight substances contained in the culture medium had beenmostly washed away together with the yeast cells into the Streamline SPunadsorbed fraction at a high efficiency and albumin was specificallyrecovered into the eluate. The HPLC pattern of the sample prepared byusing this fraction as a starting material and further purifying by theanion exchanger (DEAE) treatment showed a sharp peak of albumin (HSAmonomer) alone. Thus its purity was comparable or even superior to thesample resulted from the DEAE step of the conventional purificationprocess.

On the basis of these Test Examples, the rHSA yield resulted from theDEAE step of the conventional purification process end that resultedfrom the DEAE concentration step in the process involving the adsorbentparticle (Streamline SP) treatment step were calculated and comparedwith each other. In the process involving the adsorbent particle(Streamline SP) treatment, the number of the steps was reduced from fiveof the conventional process (i.e.,pressing→membrane→heating→membrane→cation exchanger treatment) to two.Thus the treatment time was largely shortened and the yield wasincreased by 30%.

TEST EXAMPLE 9 Analysis of Impurities Originating in Host

The culture medium of a yeast which did not produce albumin was purifiedin the same manner as the process of the present invention as describedin Example 1. Then a rabbit was immunized therewith. By using theantiserum thus obtained, testing was carried out for detectingimpurities originating in the yeast in the purified albumin solution.Enzyme immunoassay (EIA) was employed therefor. The albuminconcentration of the sample was adjusted to 250 mg/ml.

As a result, no antigenic impurity originating in the yeast was detectedfrom the purified albumin resulted after the boric acid/borate treatmentat a detection limit of 0.1 ng/ml.

TEST EXAMPLE 10 Properties of rHSA of The Present Invention Purified bythe Process of Example 1

(1) Molecular Weight The molecular weight.was determined by theabove-mentioned HPLC gel filtration method. The rHSA purified inaccordance with the process of the present invention had a molecularweight of about 67,000, i.e., almost the same as that of the HSAoriginating in plasma.

(2) Isoelectric Point

The isoelectric point was determined in accordance with the method ofAllen et al. [J. Chromatog., 146, 1 (1978)] with the use of apolyacrylamide gel. The rHSA purified in accordance with the process ofthe present invention had an isoelectric point of about 4.9, i.e.,almost the same as that of the HSA originating in plasma.

(3) Degree of Coloring

The degree of coloring was determined by using a solution of thepurified rHSA (albumin concentration: 250 mg/ml), measuring theabsorbance of this solution at 280 and 350 nm and calculating theA350/A280 ratio. The rHSA purified in accordance with the process of thepresent invention showed an extremely low degree of coloring (A350/A280)of about 0.012.

EXAMPLE 2 (1) Heating Treatment of Culture Medium

About 1,000 liter of the culture medium including cells obtained by thomethod described in EP-A-655503 in the same manner as in Example 1 washeated to 68° C. for 30 minutes as such. The heating treatment wasperformed in the presence of 10 mM of sodium caprylate. This culturemedium had a pH value of 6. Next, the heated solution was cooled toabout 25° C. and diluted about 2-fold with distilled water (totalvolume: about 2,000 liter). Then the pH value thereof was adjusted to4.5 with an acetic acid solution (99.7%) in an amount of about 1.1%(v/v) of the volume of the culture medium before dilution.

(2) Adsorbent Particle Treatment (Streamline SP Treatment)

To a Streamline SP column (C1000, 100×110 cm, gel volume: 150 liter,manufactured by Pharmacia), which had been equilibrated with a 50 mMacetate buffer (pH 4.5) containing 50 my of sodium chloride, was fedupwardly 2,000 liter of the culture medium containing the yeast cellswhich had been obtained by the above-mentioned heating treatment (1).The feeding was made at a flow rate of 100 to 250 cm/h under stirring sothat the cells might not precipitate until the addition of the culturemedium to the column was completed. Next, the same buffer (5 times byvolume as much as the column capacity) as the one employed for theequilibration of the column was fed upwardly into the column to therebywash the column at a flow rate of 100 to 500 cm/h. Subsequently, theflow direction was reversed and an eluent [a 100 mM phosphate buffer (pH9) containing 300 mM of sodium chloride, flow rate: 50 to 100 cm/h] wasfed into the column. Thus a fraction containing rHSA was obtained.

The rHSA-containing fraction thus eluted was detected by measuring theabsorbance at 280 nm.

(3) Heating Treatment

The rHSA-containing fraction thus obtained was heated at 60° C. for 1hour in the presence of 10 mM of cysteine, 10 mM of sodium caprylate,sodium oleate in an amount of 4 moles per mole of rHSA and 100 mM ofaminoguanidine hydrochloride at pH 7.5 to reduce the degree of thecoloring of rHSA and accelerate the conversion of dimer to monomer.

Table 6 shows the results of four runs using 1 ton of the culture mediumrespectively. The average yield after the heat treatment of 68° C. for30 minutes and the heat treatment with cysteine is 98.6% and 88.4%,respectively. The total yield of the four runs shows such a good resultas 37.1%, which is in well agreement with the results of Example 1 inwhich the column of the experimental scale (C50) was used. Thus, it hasbeen confirmed that the process of the present invention is reproduciblein a large scale.

TABLE 6 Degree of Run Volume rHSA Yield coloring No. Step (liter) (g)(%) (A350/A280) 1 culture medium  922 5868 100.0 0.0596 heated (68° C.,30 min) 1900 5399 92.0 0.0379 column pass 6000 — — — column eluate  200— — — heated with cysteine  62 4840 82.5 0.0268 2 culture medium  9436246 100.0 0.0547 heated (68° C., 30 min) 1960 6351 101.7 0.0375 columnpass 6400 — — — column eluate  300 — — — heated with cysteine  61 567490.9 0.0248 3 culture medium  937 6200 100.0 0.0539 heated (68° C., 30min) 1877 6261 101.1 0.0307 column pass 5777  462 7.4 — column eluate 200 — — — heated with cysteine  111 5594 90.2 0.0227 4 culture medium 916 6845 100.0 0.0520 heated (68° C., 30 min) 1885 6818 99.6 0.0556column pass 5885 — — — column eluate  300 — — — heated with cysteine 111 5804 84.8 0.0235

REFERENCE EXAMPLE 1 Determination of rHSA (Evaluation of Yield)

In the above Test Examples 1 to 8 and 10, the rHSA was quantitativelyevaluated (including the yield) by subjecting an rHSA-containingsolution to gel filtration HPLC. The detailed elution conditions are asfollows.

The solution containing rHSA was poured into a TSK-Gel G3000 SWxL column(0.78×30 cm, manufactured by Tosoh Corporation) which had beenequilibrated with a 50 mM sodium phosphate buffer (pH 6.5) containing0.1% of NaN₃ and 0.3% of NaCl. Then rHSA was eluted by using theequilibration buffer as a eluent at a flow rate of 1 ml/min ariddetermined by measuring the absorbance at 280 nm and 350 nm.

The present invention has been completed on the basis of a finding thatproteases can be easily and effectively inactivated by directly heatinga culture medium containing yeast as such. Thus, the present inventionprovides a process for easily and effectively purifying rHSA by heatinga culture medium in which yeast cells remain and contacting the heatedsolution directly with adsorbent particles suspended in a fluidized bed.Thus the number of the steps is reduced from five of the conventionalprocess (including pressing→membrane→heating→membrane→cation exchangertreatment) to two stops of heating→adsorbent particle treatment. Thusthe treatment period is largely shortened and the yield is elevated.Moreover, the problem of coloration characteristic to recombinant HSAcan be solved by the purification process of the present inventionwhereby coloring matters causing coloration can be efficientlyeliminated.

According to the purification process of the present invention,furthermore, the whole process of producing recombinant HSA includingthe steps of from the cultivation of cells to the purification can beperformed on a closed system line, thus bringing about such advantagesthat the production of HSA can be automated and that hygienicmanagement, which is essentially required in the production of rHSA as amedicine, can be easily effected.

Accordingly, the process of the present invention is highly useful as aprocess for purifying rHSA which makes it possible not only to shortenthe treatment period and to elevate the yield but also to improve thequalities of the product.

In addition, the present invention makes it possible to provide rHSAwhich does not contain impurities relating to the producer host or thelike, and which exhibits sufficiently suppressed coloration forusefulness as a drug.

While the instant invention has been described in detail and withreference to specific embodiments theoreof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed is:
 1. A process for purifying recombinant human serumalbumin comprising: (a) heating a culture medium containing said albuminand recombinant human serum albumin-producing host cells in which saidalbumin has been produced to inactivate a protease in said culturemedium; (b) contacting said heated culture medium with adsorbentparticles suspended in a fluidized bed under conditions for selectivelyadsorbing said albumin; and (c) recovering the adsorbed purifiedfraction.
 2. A process according to claim 1, for purifying recombinanthuman serum albumin, said process further comprising: (d) subjectingsaid recovered adsorbed fraction to at least one treatment selected fromthe group consisting of heat treatment in the presence of a reducingagent, hydrophobic chromatography, anion exchanger treatment, chelateresin treatment, boric acid/borate treatment and ultrafiltrationmembrane treatment.
 3. A process according to claim 1, wherein saidrecovering as recited in (c) is accomplished by reversing a flowdirection in said fluidized bed and eluting said adsorbed fraction bydownwardly feeding a buffer through bed material of said fluidized bed.4. A process according to claim 1, wherein said heating as recited in(a) is in the presence of a stabilizer.
 5. A process according to claim4, wherein said stabilizer is caprylic acid or a salt thereof.
 6. Aprocess according to claim 1, wherein said heating as recited in (a) isin the presence of aminoguanidine.
 7. A process according to claim 1,wherein said contacting as recited in (b) is in an atmosphere having anelectrical conductivity of from 0.1 to 50 mS.
 8. A process according toclaim 1, wherein the contacting as recited in (b) is performed in atleast two phases, a first phase under stirring so that the cells are notprecipitated before addition of said culture medium to said fluidizedbed is completed, and a second phase for washing said fluidized bed. 9.A process according to claim 1, wherein said process is completed usinga closed system line.